Process development for the production of mesenchymal stromal cell-derived extracellular vesicles in conventional 2D systems.

BACKGROUND: In recent years, the importance of extracellular vesicles (EVs) derived from mesenchymal stromal cells (MSCs) has increased significantly. For their widespread use, a standardized EV manufacturing is needed which often includes conventional, static 2D systems. For these system critical process parameters need to be determined. METHODS: We studied the impact of process parameters on MSC proliferation, MSC-derived particle production including EVs, EV- and MSC-specific marker expression, and particle functionality in a HaCaT cell migration assay. RESULTS: We found that cell culture growth surface and media affected MSCs and their secretory behavior. Interestingly, the materials that promoted MSC proliferation did not necessarily result in the most functional MSC-derived particles. In addition, we found that MSCs seeded at 4 × 103 cells cm-2 produced particles with improved functional properties compared to higher seeding densities. MSCs in a highly proliferative state did not produce the most particles, although these particles were significantly more effective in promoting HaCaT cell migration. The same correlation was found when investigating the cultivation temperature. A physiological temperature of 37°C was not optimal for particle yield, although it resulted in the most functional particles. We observed a proliferation-associated particle production and found potential correlations between particle production and glucose consumption, enabling the estimation of final particle yields. CONCLUSIONS: Our findings suggest that parameters, which must be defined prior to each individual cultivation and do not require complex and expensive equipment, can significantly increase MSC-derived particle production including EVs. Integrating these parameters into a standardized EV process development paves the way for robust and efficient EV manufacturing for early clinical phases.

Opportunities to debottleneck the downstream processing of the oncolytic measles virus.

Oncolytic viruses (including measles virus) offer an alternative approach to reduce the high mortality rate of late-stage cancer. Several measles virus strains infect and lyse cancer cells efficiently, but the broad application of this therapeutic concept is hindered by the large number of infectious particles required (108-1012 TCID50 per dose). The manufacturing process must, therefore, achieve high titers of oncolytic measles virus (OMV) during upstream production and ensure that the virus product is not damaged during purification by applying appropriate downstream processing (DSP) unit operations. DSP is currently a production bottleneck because there are no specific platforms for OMV. Infectious OMV must be recovered as intact, enveloped particles, and host cell proteins and DNA must be reduced to acceptable levels to meet regulatory guidelines that were developed for virus-based vaccines and gene therapy vectors. Handling such high viral titers and process volumes is technologically challenging and expensive. This review considers the state of the art in OMV purification and looks at promising DSP technologies. We discuss here the purification of other enveloped viruses where such technologies could also be applied to OMV. The development of DSP technologies tailored for enveloped viruses is necessary to produce sufficient titers for virotherapy, which could offer hope to millions of patients suffering from incurable cancer.

Innovative Optical-Sensing Technology for the Online Fouling Characterization of Silicon Carbide Membranes during the Treatment of Oily Water.

The oil and gas industry generates a large volume of contaminated water (produced water) which must be processed to recover oil before discharge. Here, we evaluated the performance and fouling behavior of commercial ceramic silicon carbide membranes in the treatment of oily wastewaters. In this context, microfiltration and ultrafiltration ceramic membranes were used for the separation of oil during the treatment of tank dewatering produced water and oily model solutions, respectively. We also tested a new online oil-in-water sensor (OMD-32) based on the principle of light scattering for the continuous measurement of oil concentrations in order to optimize the main filtration process parameters that determine membrane performance: the transmembrane pressure and cross-flow velocity. Using the OMD-32 sensor, the oil content of the feed, concentrate and permeate streams was measured continuously and fell within the range 0.0-200 parts per million (ppm) with a resolution of 1.0 ppm. The ceramic membranes achieved an oil-recovery efficiency of up to 98% with less than 1.0 ppm residual oil in the permeate stream, meeting environmental regulations for discharge in most areas.

Downstream processing of Cry4AaCter-induced inclusion bodies containing insect-derived antimicrobial peptides produced in Escherichia coli.

The Cry4AaCter tag is a pull-down tag which promotes the formation of inclusion bodies (IBs) that can be resolubilized in an alkaline buffer. Here, we used the Cry4AaCter tag to create a platform for the production of antimicrobial peptides (AMPs) in Escherichia coli featuring a uniform resolubilization process independent of the peptide fused to the pull-down tag. The Cry4AaCter tag conserves the bioactivity of fusion proteins and thus allows the purification of simple AMPs and more complex AMPs stabilized by disulfide bonds. We developed a downstream process (DSP) for the purification of IBs containing the mutated Galleria mellonella insect metalloprotease inhibitor IMPI(I38V), which has a globular structure stabilized by five disulfide bonds. IMPI(I38V) is a potent inhibitor of the M4 metalloproteases used as virulence factors by several human pathogens. We used a single crossflow filtration for the washing and resolubilization of the Cry4AaCter-induced IBs and obtained bioactive IMPI(I38V) after tag removal. We achieved a 68-fold higher protein yield using our IB system compared to an alternative DSP approach in which a GST-fusion strategy was used to produce soluble IMPI(I38V). The Cry4AaCter-based process was transferable to gloverin (another G. mellonella AMP) and the visible marker green fluorescent protein, which accumulated in fluorescent IBs, confirming it is a broadly applicable strategy for the recovery of functional proteins.

Reassessment of inclusion body-based production as a versatile opportunity for difficult-to-express recombinant proteins.

The production of recombinant proteins in the microbial host Escherichia coli often results in the formation of cytoplasmic protein inclusion bodies (IBs). Proteins forming IBs are often branded as difficult-to-express, neglecting that IBs can be an opportunity for their production. IBs are resistant to proteolytic degradation and contain up to 90% pure recombinant protein, which does not interfere with the host metabolism. This is especially advantageous for host-toxic proteins like antimicrobial peptides (AMPs). IBs can be easily isolated by cell disruption followed by filtration and/or centrifugation, but conventional techniques for the recovery of soluble proteins from IBs are laborious. New approaches therefore simplify protein recovery by optimizing the production process conditions, and often include mild resolubilization methods that either increase the yield after refolding or avoid the necessity of refolding all together. For the AMP production, the IB-based approach is ideal, because these peptides often have simple structures and are easy to refold. The intentional IB production of almost every protein can be achieved by fusing recombinant proteins to pull-down tags. This review discusses the techniques available for IB-based protein production before considering technical approaches for the isolation of IBs from E. coli lysates followed by efficient protein resolubilization which ideally omits further refolding. The techniques are evaluated in terms of their suitability for the process-scale production and downstream processing of recombinant proteins and are discussed for AMP production as an example.

High titer oncolytic measles virus production process by integration of dielectric spectroscopy as online monitoring system.

Oncolytic viruses offer new hope to millions of patients with incurable cancer. One promising class of oncolytic viruses is Measles virus, but its broad administration to cancer patients is currently hampered by the inability to produce the large amounts of virus needed for treatment (1010 -1012 virus particles per dose). Measles virus is unstable, leading to very low virus titers during production. The time of infection and time of harvest are therefore critical parameters in a Measles virus production process, and their optimization requires an accurate online monitoring system. We integrated a probe based on dielectric spectroscopy (DS) into a stirred tank reactor to characterize the Measles virus production process in adherent growing Vero cells. We found that DS could be used to monitor cell adhesion on the microcarrier and that the optimal virus harvest time correlated with the global maximum permittivity signal. In 16 independent bioreactor runs, the maximum Measles virus titer was achieved approximately 40 hr after the permittivity maximum. Compared to an uncontrolled Measles virus production process, the integration of DS increased the maximum virus concentration by more than three orders of magnitude. This was sufficient to achieve an active Measles virus concentration of > 1010 TCID50 ml-1 .

Dielectric Spectroscopy and Optical Density Measurement for the Online Monitoring and Control of Recombinant Protein Production in Stably Transformed Drosophila melanogaster S2 Cells.

The production of recombinant proteins in bioreactors requires real-time process monitoring and control to increase process efficiency and to meet the requirements for a comprehensive audit trail. The combination of optical near-infrared turbidity sensors and dielectric spectroscopy provides diverse system information because different measurement principles are exploited. We used this combination of techniques to monitor and control the growth and protein production of stably transformed Drosophila melanogaster S2 cells expressing antimicrobial proteins. The in situ monitoring system was suitable in batch, fed-batch and perfusion modes, and was particularly useful for the online determination of cell concentration, specific growth rate (µ) and cell viability. These data were used to pinpoint the optimal timing of the key transitional events (induction and harvest) during batch and fed-batch cultivation, achieving a total protein yield of ~25 mg at the 1-L scale. During cultivation in perfusion mode, the OD880 signal was used to control the bleed line in order to maintain a constant cell concentration of 5 × 107 cells/mL, thus establishing a turbidostat/permittistat culture. With this setup, a five-fold increase in productivity was achieved and 130 mg of protein was recovered after 2 days of induced perfusion. Our results demonstrate that both sensors are suitable for advanced monitoring and integration into online control strategies.

A high-throughput expression screening platform to optimize the production of antimicrobial peptides.

BACKGROUND: Antimicrobial peptides (AMPs) are promising candidates for the development of novel antibiotics, but it is difficult to produce sufficient quantities for preclinical and clinical studies due to their toxicity towards microbial expression hosts. To avoid laborious trial-and-error testing for the identification of suitable expression constructs, we have developed a small-scale expression screening platform based on a combinatorial plasmid library. RESULTS: The combinatorial library is based on the Golden Gate cloning system. In each reaction, six donor plasmids (each containing one component: a promoter, fusion partner 1, fusion partner 2, protease cleavage site, gene of interest, or transcriptional terminator) were combined with one acceptor plasmid to yield the final expression construct. As a proof of concept, screening was carried out in Escherichia coli and Pichia pastoris to study the expression of three different model AMPs with challenging characteristics, such as host toxicity or multiple disulfide bonds. The corresponding genes were successfully cloned in 27 E. coli and 18 P. pastoris expression plasmids, each in a one-step Golden Gate reaction. After transformation, small-scale expression screening in microtiter plates was followed by AMP quantification using a His6 tag-specific ELISA. Depending on the plasmid features and the expression host, the protein yields differed by more than an order of magnitude. This allowed the identification of high producers suitable for larger-scale protein expression. CONCLUSIONS: The optimization of recombinant protein production is best achieved from first principles by initially optimizing the genetic construct. The unrestricted combination of multiple plasmid features yields a comprehensive library of expression strains that can be screened for optimal productivity. The availability of such a platform could benefit all laboratories working in the field of recombinant protein expression.

The components of shear stress affecting insect cells used with the baculovirus expression vector system.

Insect-based expression platforms such as the baculovirus expression vector system (BEVS) are widely used for the laboratory- and industrial-scale production of recombinant proteins. Thereby, major drawbacks to gain high-quality proteins are the lytic infection cycle and the shear sensitivity of infected insect cells due to turbulence and aeration. Smaller bubbles were formerly assumed to be more harmful than larger ones, but we found that cell damage is also dependent on the concentration of protective agents such as Pluronic®. At the appropriate concentration, Pluronic forms a layer around air bubbles and hinders the attachment of cells, thus limiting the damage. In this context, we used microaeration to vary bubble sizes and confirmed that size is not the most important factor, but the total gas surface area in the reactor is. If the surface area exceeds a certain threshold, the concentration of Pluronic is no longer sufficient for cell protection. To investigate the significance of shear forces, a second study was carried out in which infected insect cells were cultivated in a hollow fiber module to protect them from shear forces. Both model studies revealed important aspects of the design and scale-up of BEVS processes for the production of recombinant proteins.

Effect of prebiotics of Agave salmiana fed to healthy Wistar rats.

BACKGROUND: Inulin and other fructans are synthesized and stored in mezcal agave (Agave salmiana). Fructans provide several health benefits and have excellent technological properties, but only few data report their physiological effect when added in the diet. RESULTS: Here, we studied the physiological effects of fructans obtained from A. salmiana when added in the diet of Wistar rats. Results showed favorable changes on Wistar rats when the fructans was added to their diet, including the decrease of the pH in the feces and the increase of the number of lactic acid bacteria (CFU g-1 ) (Lactobacillus spp. and Bifidobacterium spp.), even these changes were enhanced with the synbiotic diet (fructans plus B. animalis subsp. lactis). Synbiotic diet, developed changes in the reduction of cholesterol and triglycerides concentrations in serum, with statistical differences (P < 0.05). Histological analysis of colon sections showed that synbiotic diet promoted colon cells growth suggesting that fructans from A. salmiana confer beneficial health effects through gut microbiota modulation. CONCLUSION: Our data underline the advantage of targeting the gut microbiota by colonic nutrients like specific structure of fructans from A. salmiana, with their beneficial effects. More studies are necessary to define the role of fructans to develop more solid therapeutic solutions in humans. © 2016 Society of Chemical Industry.

The potential of the Galleria mellonella innate immune system is maximized by the co-presentation of diverse antimicrobial peptides.

Antimicrobial peptides (AMPs) are ubiquitous components of the insect innate immune system. The model insect Galleria mellonella has at least 18 AMPs, some of which are still uncharacterized in terms of antimicrobial activity. To determine why G. mellonella secretes a repertoire of distinct AMPs following an immune challenge, we selected three different AMPs: cecropin A (CecA), gallerimycin and cobatoxin. We found that cobatoxin was active against Micrococcus luteus at a minimum inhibitory concentration (MIC) of 120 µm, but at 60 µm when co-presented with 4 µm CecA. In contrast, the MIC of gallerimycin presented alone was 60 µm and the co-presentation of CecA did not affect this value. Cobatoxin and gallerimycin were both inactive against Escherichia coli at physiological concentrations, however gallerimycin could potentiate the sublethal dose of CecA (0.25 µm) at a concentration of 30 µm resulting in 100% lethality. The ability of gallerimycin to potentiate the CecA was investigated by flow cytometry, revealing that 30 µm gallerimycin sensitized E. coli cells by inducing membrane depolarization, which intensified the otherwise negligible effects of 0.25 µm CecA. We therefore conclude that G. mellonella maximizes the potential of its innate immune response by the co-presentation of different AMPs that become more effective at lower concentrations when presented simultaneously.

An Innovative Optical Sensor for the Online Monitoring and Control of Biomass Concentration in a Membrane Bioreactor System for Lactic Acid Production.

Accurate real-time process control is necessary to increase process efficiency, and optical sensors offer a competitive solution because they provide diverse system information in a noninvasive manner. We used an innovative scattered light sensor for the online monitoring of biomass during lactic acid production in a membrane bioreactor system because biomass determines productivity in this type of process. The upper limit of the measurement range in fermentation broth containing Bacillus coagulans was ~2.2 g·L(-1). The specific cell growth rate (µ) during the exponential phase was calculated using data representing the linear range (cell density ≤ 0.5 g·L(-1)). The results were consistently and reproducibly more accurate than offline measurements of optical density and cell dry weight, because more data were gathered in real-time over a shorter duration. Furthermore, µ(max) was measured under different filtration conditions (transmembrane pressure 0.3-1.2 bar, crossflow velocity 0.5-1.5 m·s(-1)), showing that energy input had no significant impact on cell growth. Cell density was monitored using the sensor during filtration and was maintained at a constant level by feeding with glucose according to the fermentation kinetics. Our novel sensor is therefore suitable for integration into control strategies for continuous fermentation in membrane bioreactor systems.

Large-Scale Expansion of Human Liver Stem Cells Using Two Different Bioreactor Systems.

The assessment of human liver stem cells (HLSCs) as cell therapeutics requires scalable, controlled expansion processes. We first focused on defining appropriate process parameters for HLSC expansion such as seeding density, use of antibiotics, optimal cell age and critical metabolite concentrations in conventional 2D culture systems. For scale-up, we transferred HLSC expansion to multi-plate and stirred-tank bioreactor systems to determine their limitations. A seeding density of 4000 cells cm-2 was needed for efficient expansion. Although growth was not significantly affected by antibiotics, the concentrations of lactate and ammonia were important. A maximum expansion capacity of at least 20 cumulative population doublings (cPDs) was observed, confirming HLSC growth, identity and functionality. For the expansion of HLSCs in the multi-plate bioreactor system Xpansion (XPN), the oxygen supply strategy was optimized due to a low kLa of 0.076 h-1. The XPN bioreactor yielded a final mean cell density of 94 ± 8 × 103 cells cm-2, more than double that of the standard process in T-flasks. However, in the larger XPN50 device, HLSC density reached only 28 ± 0.9 × 103 cells cm-2, while the glucose consumption rate increased 8-fold. In a fully-controlled 2 L stirred-tank bioreactor (STR), HLSCs expanded at a comparable rate to the T-flask and XPN50 processes in a homogeneous microenvironment using advanced process analytical technology. Ultimately, the scale-up of HLSCs was successful using two different bioreactor systems, resulting in sufficient numbers of viable, functional and undifferentiated HLSCs for therapeutic applications.

Identification of transglutaminase substrates from porcine nucleus pulposus as potential amplifiers in cross-linking cell scaffolds.

Nucleus pulposus from the porcine intervertebral disc was separated chromatographically to discover substrates of microbial transglutaminase. Highly purified proteins were prepared, among them type II collagen, the major protein of the nucleus pulposus. Determination of substrates was performed by transglutaminase-mediated incorporation of biotinylated probes displaying several glutamine and lysine donor proteins. Type II collagen was only labeled if smaller nucleus pulposus proteins were present. One of the modulating proteins was serotransferrin, a lysine donor substrate of bacterial transglutaminase. An additional substrate was the carboxy-terminal propeptide of type II collagen, chondrocalcin. Chondrocalcin, a regulator of type II collagen fibrillogenesis, occurs abundantly in juvenile cartilage and nucleus pulposus. Accordingly, the protein may be regarded as an excellent additive for the preparation of injectable stem cells in nucleus pulposus-like matrices cross-linked by microbial transglutaminase.

A Bioreactor-Based Yellow Fever Virus-like Particle Production Process with Integrated Process Analytical Technology Based on Transient Transfection.

Yellow Fever (YF) is a severe disease that, while preventable through vaccination, lacks rapid intervention options for those already infected. There is an urgent need for passive immunization techniques using YF-virus-like particles (YF-VLPs). To address this, we successfully established a bioreactor-based production process for YF-VLPs, leveraging transient transfection and integrating Process Analytical Technology. A cornerstone of this approach was the optimization of plasmid DNA (pDNA) production to a yield of 11 mg/L using design of experiments. Glucose, NaCl, yeast extract, and a phosphate buffer showed significant influence on specific pDNA yield. The preliminary work for VLP-production in bioreactor showed adjustments to the HEK cell density, the polyplex formation duration, and medium exchanges effectively elevated transfection efficiencies. The additive Pluronic F-68 was neutral in its effects, and anti-clumping agents (ACA) adversely affected the transfection process. Finally, we established the stirred-tank bioreactor process with integrated dielectric spectroscopy, which gave real-time insight in relevant process steps, e.g., cell growth, polyplex uptake, and harvest time. We confirmed the presence and integrity of YF-VLP via Western blot, imaging flow cytometry measurement, and transmission electron microscopy. The YF-VLP production process can serve as a platform to produce VLPs as passive immunizing agents against other neglected tropical diseases.

Identification and characterisation of the tegument-expressed aldehyde dehydrogenase SmALDH_312 of Schistosoma mansoni, a target of disulfiram.

Schistosomiasis is an infectious disease caused by blood flukes of the genus Schistosoma and affects approximately 200 million people worldwide. Since Praziquantel (PZQ) is the only drug for schistosomiasis, alternatives are needed. By a biochemical approach, we identified a tegumentally expressed aldehyde dehydrogenase (ALDH) of S. mansoni, SmALDH_312. Molecular analyses of adult parasites showed Smaldh_312 transcripts in both genders and different tissues. Physiological and cell-biological experiments exhibited detrimental effects of the drug disulfiram (DSF), a known ALDH inhibitor, on larval and adult schistosomes in vitro. DSF also reduced stem-cell proliferation and caused severe tegument damage in treated worms. In silico-modelling of SmALDH_312 and docking analyses predicted DSF binding, which we finally confirmed by enzyme assays with recombinant SmALDH_312. Furthermore, we identified compounds of the Medicine for Malaria Venture (MMV) pathogen box inhibiting SmALDH_312 activity. Our findings represent a promising starting point for further development towards new drugs for schistosomiasis.

Screening for Biofilm-Stimulating Factors in the Freshwater Planctomycete Planctopirus limnophila to Improve Sessile Growth in a Chemically Defined Medium.

Planctomycetes such as Planctopirus limnophila offer a promising source of bioactive molecules, particularly when they switch from planktonic to sessile growth, but little is known about the corresponding biosynthetic gene clusters and how they are activated. We therefore screened for factors that promote sessile growth and biofilm formation to enable the cultivation of P. limnophila in a fixed-bed reactor. We carried out screening in microtiter plates focusing on biofilm formation and changes in optical density in response to various C:N ratios, metal ions, and oxidative stress. We used MTT assays and crystal violet staining to quantify biofilm formation. Positive factors were then validated in a fixed-bed bioreactor. The initial screen showed that D1ASO medium supplemented with NH4Cl to achieve a C:N ratio of 5.7:1, as well as 50 µM FeSO4 or CuSO4, increased the biofilm formation relative to the control medium. Exposure to H2O2 did not affect cell viability but stimulated biofilm formation. However, the same results were not replicated in the fixed-bed bioreactor, probably reflecting conditions that are unique to this environment such as the controlled pH and more vigorous aeration. Although we were able to cultivate P. limnophila in a fixed-bed bioreactor using a chemically defined medium, the factors that stimulate biofilm formation and inhibit planktonic growth were only identified in microtiter plates and further evaluation is required to establish optimal growth conditions in the bioreactor system.

A New Control Strategy for High-Pressure Homogenization to Improve the Safety of Injectable Lipid Emulsions.

Intravenous lipid emulsions are biocompatible formulations used as clinical nutrition products and lipid-based delivery systems for sparingly soluble drugs. However, the particle-size distribution is associated with risks of embolism. Accordingly, the mean particle diameter (MPD) and particle-distribution tailing (characterized as the pFAT5 value) are critical quality attributes that ensure patient safety. Compliance with the limits stated in the United States Pharmacopoeia is ensured by high-pressure homogenization, the final step of the manufacturing process. The US Food and Drug Administration's Quality-by-Design approach requires a control strategy based on deep process understanding to ensure that products have a consistent and predefined quality. Here we investigated the process parameters of a jet-valve high-pressure homogenizer, specifically their effect on the MPD, pFAT5 value and droplet count (determined by microscopy) during the production of a Lipofundin MCT/LCT 20% formulation. We provide deep insight into droplet breakup and coalescence behavior when varying the process pressure, emulsion temperature and number of homogenization cycles. We found that high shear forces are not required to reduce the pFAT5 value of the particle distribution. Finally, we derived a control strategy for a rapid and cost-efficient two-cycle process that ensures patient safety over a large control space.

Statistical experimental designs to optimize the transient transfection of HEK 293T cells and determine a transfer criterion from adherent cells to larger-scale cell suspension cultures.

The transient transfection of mammalian cells is a rapid and versatile platform for the manufacture of recombinant proteins, but industrial processes depend on reliable scalability and efficient conversion from adherent to suspension cell cultures. Here we describe the optimized transfection of HEK 293T cells in both culture formats. DMEM was the best transfection medium for adherent HEK 293T cells, so we determined the kinetics of linear polyethyleneimine (LPEI) polyplex formation with plasmid DNA (pDNA) and subsequent cellular uptake. Statistical experimental designs revealed optimal transfection efficiency using 0.7 pg pDNA and 4.5 pg LPEI per cell. We used the amount of pDNA and LPEI per cell as the transfer criterion for HEK 293T/17 SF cell suspension cultures in FreeStyle 293 medium and confirmed optimal transfection at 1.1 pg pDNA and 6.6 pg LPEI per cell. We observed a strong correlation between polyplex size, transfection efficiency and post-transfection cell viability. Suspension cell transfection could be scaled to a 100-mL working volume without loss of efficiency. We conclude that pg pDNA and pg LPEI per cell is a suitable transfer criterion allowing the optimization of transient transfection using statistical experimental designs, thus minimizing the amount of pDNA and LPEI used without sacrificing transfection efficiency.

A Combined Ultrafiltration/Diafiltration Process for the Purification of Oncolytic Measles Virus.

Measles virus (MV) is an important representative of a new class of cancer therapeutics known as oncolytic viruses. However, process intensification for the downstream purification of this fragile product is challenging. We previously found that a mid-range molecular weight cut-off (300 kDa) is optimal for the concentration of MV. Here, we tested continuous and discontinuous diafiltration for the purification of MV prepared in two different media to determine the influence of high and low protein loads. We found that a concentration step before diafiltration improved process economy and MV yield when using either serum-containing or serum-free medium. We also found that discontinuous diafiltration conferred a slight benefit in terms of the permeate flow, reflecting the repetitive dilution steps and the ability to break down parts of the fouling layer on the membrane. In summary, the combined ultrafiltration/diafiltration process is suitable for the purification of MV, resulting in the recovery of ~50% infectious virus particles with a total concentration factor of 8 when using 5 diavolumes of buffer.